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Methods S1
Random Amplification of Polymorphic DNA (RAPD). H. pylori genomic DNA was
PCR-amplified in 25 μl reaction mix containing 12.5 ul Premix Ex-Taq (Takara Bio
Inc.), 20 pmoles of primer and 200 ng of template DNA. The parameters set in a Perkin
Elmer thermocycler 2400 (PE Applied Biosystems, Foster City) were: [94oC, 5 min;
36oC, 5 min; and 72oC, 5 min], 4 cycles, followed by 30 cycles of [94oC, 1 min; 36oC, 1
min; and 72oC, 2 min], and finally 72oC for 10 min. PCR products were resolved by
electrophoresis in ethidium bromide-stained 2% agarose gels.
16S rRNA gene amplification and sequencing. Total DNA was extracted from each
patient’s isolate and PCR-amplified using published primers [1]. Amplification was
performed at annealing temperature of 50oC for 35 cycles using a Perkin Elmer 2400
thermocycler. The amplified segment was resolved by electrophoresis on ethidium
bromide stained-agarose gel and visualized by UV transillumination. The DNA band was
cut out from the agarose gel and purified using MinEluteTM Gel extraction kit (Qiagen).
The sequence of the purified 16S rRNA gene was determined using sets of overlapping
primers [1], a dye terminator reagent and an automated DNA sequencer (PE Applied
Biosystems, Foster City).
In situ hybridization
1. Pre-hybridization: Sections were deparaffinized, rehydrated in grades of ethanol, and
treated with proteinase K (Roche Diagnostic, Indianapolis) for unmasking nucleic acids
and preparation for probe penetration. Sections were treated with hybridization mixture
and incubated with bovine serum albumin (BSA) to block non-specific binding.
2. Hybridization: 50-100 μl of the probes denatured by heating for 10 min at 65ºC were
placed on the tissue and incubated for 18 hrs at 37ºC.
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3. Post-hybridization: unbound probe was removed by successive washes
4. Detection of H. pylori Gene Expression. Sections were incubated with streptavidin
conjugated alkaline phosphatase (AP; Roche Diagnostic, Indianapolis) or avidin-labeled
peroxidase to detect the biotin-labeled 16S rRNA probe.
Unbound antibody-alkaline phosphatase peroxidase were washed away, bound alkaline
phosphatase or peroxidase was detected using a chromogenic substrate (nitroblue
tetrazolium (BCIP/NBT) or 3,3’ diaminobenzidine (DAB) respectively), and slides were
counterstained (Nuclear Fast Red or hematoxylin QS for alkaline phosphatase or
peroxidase detection use respectively), washed, dehydrated, cleared in xylene, and
mounted with Permount (Biomedia Corporation, Foster City).
For fluorescence ISH (FISH), a similar method was used [labeling of H. pylori 16S rRNA
probe was labeled with biotin, detection with avidin-fluorescein isothiocyanate (FITC),
mounting with Vectashield (Vector), resulting in bright green reaction] [2,3].
Reference List
1. Eckloff BW, Podzorski RP, Kline BC, Cockerill FR3 (1994) A comparison of 16S
ribosomal DNA sequences from five isolates of Helicobacter pylori. Int J
Syst Bacteriol 44: 320-323.
2. Semino-Mora C, Doi SQ, Marty A, Simko V, Carlstedt I, Dubois A (2003)
Intracellular and interstitial expression of H. pylori virulence genes in
gastric precancerous intestinal metaplasia and adenocarcinoma. J Infect Dis
187: 1165-1177.
3. Aspholm M, Kalia A, Ruhl S, Schedin S, Arnqvist A, et al. (2006) Helicobacter
pylori adhesion to carbohydrates. In: Minoru Fukuda, editors. Methods in
Enzymology. New York: Elsevier. pp. 293-239.